Small molecule compositions for binding to hsp90

ABSTRACT

Structural differences in binding pockets of members of the HSP90 family can be exploited to achieve differential degradation of kinases and other signaling proteins through the use of designed small molecules which interact with the N-terminal binding pocket with an affinity which is greater than ADP and different from the ansamycin antibiotics for at least one species of the HSP90 family. Moreover, these small molecules can be designed to be soluble in aqueous media, thus providing a further advantage over the use of ansamycin antibiotics. Pharmaceutical compositions can be formulated containing a pharmaceutically acceptable carrier and a molecule that includes a binding moiety which binds to the N-terminal pocket of at least one member of the HSP90 family of proteins. Such binding moieties were found to have antiproliferative activity against tumor cells which are dependent on proteins requiring chaperones of the HSP90 family for their function. Different chemical species have different activity, however, allowing the selection of, for example Her2 degradation without degradation of Raf kinase. Thus, the binding moieties possess an inherent targeting capacity. In addition, the small molecules can be linked to targeting moieties to provide targeting of the activity to specific classes of cells. Thus, the invention further provides a method for treatment of diseases, including cancers, by administration of these compositions. Dimeric forms of the binding moieties may also be employed.

This application claims the benefit of U.S. Provisional Application No.60/245,177, filed Nov. 2, 2000, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

This application relates to small molecules that bind to the HSP90family of proteins, and to methods for making and using such smallmolecules.

The HSP90 family of proteins has four recognized members in mammaliancells: Hsp90 α and β, Grp94 and Trap-1. Hsp90 α and β exist in thecytosol and the nucleus in association with a number of other proteins.Hsp90 is the most abundant cellular chaperone, and has been shown inexperimental systems to be required for ATP-dependent refolding ofdenatured or “unfolded”proteins. It has therefore been proposed tofunction as part of the cellular defense against stress. When cells areexposed to heat or other environmental stresses, the aggregation ofunfolded proteins is prevented by pathways that catalyze their refoldingor degradation. This process depends on the association of the unfoldedprotein in an ordered fashion with multiple chaperones (Hsp 60, 90 and70 and p23), forming a “refoldosome” and ultimately the ATP-dependentrelease of the chaperones from the refolded protein.

Hsp90 may also play a role in maintaining the stability and function ofmutated proteins. It seems to be required for expression of mutated p53and v-src to a much greater extent than for their wild-typecounterparts. It has been suggested that this occurs as a result ofhsp90-mediated suppression of the phenotypes of mutations that lead toprotein unfolding.

Hsp90 is also necessary to the conformational maturation of several keyproteins involved in the growth response of the cell to extracellularfactors. These include the steroid receptors as well as certaintransmembrane kinases (i.e., Raf serine linase, v-src and Her2). Themechanism whereby hsp90 affects these proteins is not fully understood,but appears to be similar to its role in protein refolding. In the caseof the progesterone receptor, it has been shown that binding and releaseof hsp90 from the receptor occurs in a cyclic fashion in concert withrelease of other chaperones and immunophilins and is required for highaffinity binding of the steroid to the receptor. Thus, hsp90 couldfunction as a physiologic regulator of signaling pathways, even in theabsence of stress.

Hsp90 has also been shown to be overexpressed in multiple tumor typesand as a function of oncogenic transformation. Whether it plays anecessary role in maintaining transformation is unknown, but it couldhave at least three functions in this regard. Cancer cells grow in anenvironment of hypoxia, low pH and low nutrient concentration. They alsorapidly adapt to or are selected to become resistant to radiation andcytotoxic chemotherapeutic agents. Thus, the general role of hsp90 inmaintaining the stability of proteins under stress may be necessary forcell viability under these conditions. Secondly, cancer cells harbormutated oncogenic proteins. Some of these are gain-of-function mutationswhich are necessary for the transformed phenotype. Hsp90 may be requiredfor maintaining the folded, functionally-active conformation of theseproteins. Thirdly, activation of signaling pathways mediated by steroidreceptors, Raf and other hsp90 targets is necessary for the growth andsurvival of many tumors which thus probably also require functionalhsp90.

These characteristics of hsp90 make it a viable target for therapeuticagents. HSP90 family members possess a unique pocket in their N-terminalregion that is specific to and conserved among all hsp90s from bacteriato mammals, but which is not present in other molecular chaperones. Theendogenous ligand for this pocket is not known, but it binds ATP and ADPwith low affinity and has weak ATPase activity. In addition, theansamycin antibiotics geldanamycin (GM) and herbimycin (HA) have beenshown to bind to this conserved pocket. This binding affinity has beenshown for all members of the HSP90 family. International PatentPublication No. WO98/51702, which is incorporated herein by reference,discloses the use of ansamycin antibiotics coupled to a targeting moietyto provide targeted delivery of the ansamycin leading to the degradationof proteins in and death of the targeted cells. International PatentPublication No. WO00/61578 relates to bifunctional molecules having twomoieties which interact with the chaperone protein hsp90, including inparticular homo- and heterodimers of ansamycin antibiotics. Thesebifunctional molecules act to promote degradation and/or inhibition ofHER-family tyrosine kinases and are effective for treatment of cancerswhich overexpress Her-kinases.

While the use of geldanamycin and other ansamycin antibiotics and theirderivatives provides for effective degradation of a number of kinasesand other signaling proteins, they generally lack significantselectivity, and are instead effective to degrade a broad spectrum ofproteins. This can increase the risk of undesirable side effects.Furthermore, anasmycin antibiotics are insoluble or at best poorlysoluble in aqueous media. This complicates administration. Thus, thereremains room for improvement of therapeutic agents that bring about thedegradation of kinases and other signaling proteins via interaction withmembers of the HSP90 family of chaperone proteins.

SUMMARY OF THE INVENTION

While the members of the HSP90 family of proteins are characterized by aunique N-terminal binding pocket that is highly conserved, there arestructural differences between the pockets of the various members. Ithas now been found that these structural differences can be exploited toachieve differential degradation of kinases and other signaling proteinsthrough the use of designed small molecules which interact with theN-terminal binding pocket with an affinity which is greater than ADP anddifferent from the ansamycin antibiotics. Moreover, these smallmolecules can be designed to be soluble in aqueous media, thus providinga further advantage over the use of ansamycin antibiotics.

The N-terminal pockets of the HSP90 of family of proteins contain fivepotential binding sites. In the case of hsp90 α, these binding sitesare:

-   (a) a top binding site comprising Lys112,-   (b) a second top binding site comprising Lys58, Asp54, Phe138    backbone, Gly 135 and Asn106;-   (c) a bottom binding site comprising Asp93, Ser52 and Asn51;-   (d) a hydrophobic lateral binding site comprising Val150, Met98,    Val186, Phe138, Leu107, Leu103, Val186 and Trp162; and-   (e) a small hydrophobic bottom binding site comprising Thr184,    Val186, Val150 and Ile91.    The present invention provides pharmaceutical compositions    comprising a pharmaceutically acceptable carrier and a molecule    comprising a binding moiety which binds to the N-terminal pocket of    at least one member of the HSP90 family of proteins. This binding    moiety interacts with the N-terminal pocket with an affinity greater    than ADP but less than geldanamycin for at least one specifies    protein in the HSP90 family. Further, the binding moiety has a    backbone which can achieve a folded C-configuration when disposed    within the N-terminal pocket of a member of the HSP90 family of    proteins. The binding moiety also has substituents on the backbone    directed in orientations to interact with a plurality of the    potential binding sites within the N-terminal pocket.

The binding moieties of the invention were found to haveantiproliferative activity against tumor cells which are dependent onproteins requiring chaperones of the HSP90 family for their function.Different chemical species have different activity, however, allowingthe selection of, for example Her2 degradation without degradation ofRaf kinase. Thus, the binding moieties of the invention possess aninherent targeting capacity. In addition, the small molecules can belinked to targeting moieties to provide targeting of the activity tospecific classes of cells. Thus, the invention further provides a methodfor treatment of diseases, including cancers, by administration of thesecompositions. Dimeric forms of the binding moieties may also beemployed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows aligned sequences of amino acids contributing to theN-terminal binding pocket in known members of the HSP90 family ofchaperone proteins, Hsp90 α (Seq. ID No. 1), GRP94 (Seq. ID. No. 2),Hsp90 β (Seq. ID No. 3) and Trap1 (Seq. ID. No. 4);

FIG. 2 shows a three dimensional drawings of the pocket with a bindingmoiety of the invention disposed therein;

FIGS. 3A and B show the conformation of PU3 inside the hsp90 pocket, asdetermined by molecular modeling, and outside of the pocket asdetermined by x-ray crystallography;

FIG. 4 shows an exemplary binding moiety/compound based on a purinenucleus, and indicates the interactions of the various substituents withbinding sites in the N-terminal pocket of members of the HSP90 family;

FIG. 5 shows the structure of compounds in accordance with theinvention;

FIG. 6A shows a synthetic procedure for making compounds in accordancewith the invention;

FIG. 6B shows exemplary 9-N alkylation compounds that were tested foractivity. As reflected in Table 1, not all of the tested compounds wereactive, and some of the compounds could not be tested for activitybecause they were insoluble.

FIG. 7A shows a synthetic scheme for introduction of functional groupsat the X3 position for interaction with the small hydrophobic bindingsite;

FIG. 7B shows a second synthetic scheme for introduction of functionalgroups at the X3 position for interaction with the small hydrophobicbinding site;

FIGS. 8A and B show synthetic schemes for introduction of variations atthe X2 site;

FIG. 9 shows a synthetic scheme for production of binding moieties inaccordance with the invention;

FIG. 10 shows a synthetic scheme for introducing variations in thelength and character of the bridge between the purine and the phenylgroups in PU family compounds;

FIG. 11 shows dimerization or the addition of targeting moieties;

FIG. 12 shows a schematic methodology for assessing comparativeaffinities of binding moieties;

FIG. 13 shows the specific structures of PU1-PU4;

FIGS. 14A-C show the antiproliferative effect of PU3 on breast cancercell lines;

FIG. 15 shows X₁ substituents of compounds tested in one example;

FIGS. 16A and B show a procedure for modifying PU3 and usingbiotinylation for immobilization;

FIGS. 17A and B show degradation of Akt protein by PU3 and PU24FC1;

FIGS. 18 A and B show the correlation between growth arrest, Her2 totalprotein degradation and Hsp90 binding efficacy; and

FIG. 19 shows Her2 degradation with PU24FC1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to small molecules which bindpreferentially and with an affinity greater than ADP and less thangeldanamycin to the N-terminal pocket of one or more members of theHSP90 family of proteins, for example, to one or more of hsp90 α or β,Grp94 and Trap-1, or proteins with similar pockets, for example DNAgyrase and MutL. As used in the specification and claims of thisapplication, the term “N-terminal pocket of HSP90” refers to the pocketto which geldanamycin and other ansamycin antibiotics bind, and which isoccupied by ATP/ADP.

FIG. 1 shows aligned sequences comparing the structures of each of theknown members of the HSP90 family of proteins. In the case of hsp90 α,the five potential binding sites are:

-   (a) a top binding site comprising Lys112;-   (b) a second top binding site comprising Lys58, Asp54, Phe138    backbone, Gly 135 and Asn106;-   (c) a bottom binding site comprising Asp93, Ser52 and Asn51;-   (d) a hydrophobic lateral binding site comprising Val150, Met98,    Val186, Phe138, Leu107, Leu103, Val186 and Trp162; and-   (e) a small hydrophobic bottom binding site comprising Thr184,    Val186, Val150 and Ile91.    As indicated in FIG. 1, the N-terminal pocket, while highly    conserved in HSP90 family of proteins, does have differences between    the various members. The binding moieties of the invention exploit    these differences to provide compositions which afford differential    degradation of kinases and other signaling proteins. As used in the    specification and claims of this application, the term “differential    degradation” refers to either degradation of one kinase or signaling    protein to a greater extent than another kinase or signaling    protein, where both would be degraded in the presence of    geldanamycin, or to the degradation of a kinase or signaling protein    to a different product than would be obtained in the presence of    geldanamycin.

The size and shape of the N-terminal binding pocket are described inStebbins et al., “Crystal Structure of an Hsp90-Geldanamycin Complex:Targeting of a Protein Chaperone by an Antitumor Agent” Cell 89: 239-250(1997). There it is observed that “the pocket can be described as aflat-bottomed cone; it is about 15 Å deep, 12 Å in diameter near itssurface, 8 Å midway down, and wide enough at the bottom to hold threewater molecules. The binding moieties in the pharmaceutical compositionsof the invention are designed to fit within this pocket, and to interactwith a plurality of the potential binding sites within the N-terminalpocket.

FIG. 2 shows a three dimensional drawing of the pocket with a bindingmoiety of the type shown in FIG. 4 disposed therein to assist invisualizing the molecular design process. FIGS. 3A and B show theconformation of a designed binding moiety based on a purine nucleus(PU3) inside the hsp90 pocket, as determined by x-ray crystallography,inside the pocket, and without the pocket shown, respectively. Theopening 10 of the pocket is disposed near the bottom of the figure. Thepocket itself has four arms, arm 11 which is nearest the opening 10 andwhich include the bottom binding site. As reflected in the FIG. 4, theinterior of this arm interacts with the amino substituent of the purinenucleus.

Second arm 13 connects to the first arm at an angle of about 95-110° C.Third arm 12 connects to the first arm 11 at an angle of about 100-120°.These two arms contain the two binding sites and are generallyhydrophilic in nature. The substituent X₂ (FIG. 4) fits within one ofthese two arms, resulting in the bending of the binding moiety into thecharacteristic C-shape. The ability to adopt this C-shape must be takeninto account when selecting molecules to act as binding moieties. Asnoted in FIG. 2, the separation between arms 11 and 13 (9.5 to 11 Å) isgreater than the separation between arms 11 and 12 (8-9 Å). Thus,selection of a longer substituent (X₂) may favor insertion of themolecule into arm 13 over arm 12. The fourth arm 14 contains the lateralhydrophobic pocket, and receives the substituent X₁ (FIG. 4 ). Thispocket has a volume of ≈100 Å³ and is generally hydrophobic incharacter. Thus, the substituent X₁ (FIG. 4 ) is selected to fill thispocket and to interact with the hydrophobic residues.

In a first embodiment of the invention, the binding moiety is a completemolecule. FIG. 4 shows an exemplary structure of this type based on apurine nucleus, and indicates the interactions between the substituentsand the binding sites within the N-terminal binding pocket. In FIG. 4,the substituent X₁ may be any hydrophobic chain (linear, branchedaliphatic, aromatic, acyclic or cyclic, containing C, H, N, O and/or Satoms that fits within the pocket); X₂ is from 1 to five hydrogenacceptor functionalities such as OR, OCOR, NCOR and the like that fitswithin the pocket and optionally plus an electron donating group able toenhance the activity of such groups, such as halogen, and X₃ is anysmall size substituent to fit within the pocket, for example a groupsuch as fluorine. In the embodiment of the invention, a structuraldifference between the molecules of the invention and geldanamycinantibiotics and derivatives is the interaction between these bindingmoieties and the hydrophobic lateral binding site and small hydrophobicbinding site. As observed in Stebbins et al., these sites are located atthe bottom of the pocket, and geldanamycin does not fill these portions.Rather, the methyl group of geldanamycin extends only partly into thelateral pocket, leaving room at the bottom of the pocket for watermolecules. In contrast, in compositions of the invention, thesubstituents may be selected to fill at least the hydrophobic lateralbinding site in the pocket. As used in this application, the term “fill”refers to the occupancy of a binding site to an extent such that thereis not room remaining to accommodate a water molecule.

Additional examples of binding moieties within the scope of theinvention that are designed to fit within the N-terminal pocket of HSP90proteins and interact with the pockets with affinity between that of ADPand geldanamycin for at least one member of the HSP90 family are shownin FIG. 5. In these compositions, X₁ is a hydrophobic chain (linear,branched aliphatic, aromatic, acyclic or cyclic, containing C, H, N, Oand/or S atoms that first within the pocket), or COR, where R is ahydrophobic chain; X₂ is from 1 to 5 hydrogen acceptor/donorfunctionalities, which may be the same or different; X₃ is a small sizegroup such as alkyl (saturated, unsaturated, cyclic or linear), alkoxy(for example methoxy or ethoxy), halogen or SR (where R is methyl orethyl); X₄ is H or as X₃, X₅ is H or as X₁; X₆ is —NH₂, —OR (R being Hor alkyl), or —CONH₂ or a similar hydrogen donor functionality and R₁ isH or alkyl. These molecules are all capable for folding into a C-shapedconfiguration and interact with at least three of the potential bindingsites, including the hydrophobic lateral site. It will be appreciatedthat other compounds with different nuclei but comparable overall volume(˜200-500 Å³) and dimensions can be designed in the same manner and fallwithin the scope of the present invention.

The compositions having structures indicated as the PU family in FIG. 5can be synthesized using the procedure outlined in FIG. 6A. A carboxylicacid starting material is converted to an acid fluoride by reaction withcyanuric fluoride in pyridine/DCM or to an acid chloride by reactingwith SOCl₂. This acid halide is reacted with an amino-substitutedpyrimidine (such as 4,5,6-triaminopyrimidine sulfate in DIEA/DMF or2,4,5,6-tetraaminopyrimidine sulfate in aqueous NaOH, or either sulfatewith K₂CO₃ in DMF) to produce an intermediate product (PY-A, PY-B, PY-Cin FIG. 1) which undergoes ring closure to produce substituted 8-benzylpurine derivatives (PU-A, PU-B, PU-C) which are useful compositions inaccordance with the invention. If desired, a alkylation reaction, suchas a Mitsonobu alkylation, may be performed to add an alkyl group, R,(with or without functional substituents) to the nitrogen at the9-position. Exemplary compounds made by 9-N alkylation of the PU familyprecursor PU-C are shown in FIG. 6B.

In some embodiments of the invention, the compound has the formula

wherein Y is CH, O, N or O—CH,

X₁ is the substituent formed by removing the OH from an alcohol fromFIG. 6B selected from the group consisting of

X₂ is from one to five non-hydrogen groups independently selected fromthe group consisting of halogen and methoxy, X₃ is halogen, X₄ is absentwhen Y is O or hydrogen, halogen, alkyl, alkoxy, or —SCH₃ or —SCH₂CH₃,and X₆ is —NH₂, —OH, —O-Alkyl, or —CONH₂.

FIGS. 7A and B show synthetic schemes for introduction of functionalgroups at the X3 position for interaction with the small hydrophobicbinding site. FIGS. 8A and B show synthetic schemes for introduction ofvariations at the X2 site.

FIG. 9 shows a synthetic scheme for synthesis of the RD class ofcompounds from FIG. 5. Other classes shown in FIG. 5 can be made usingcomparable synthetic approaches.

The compositions of the invention may be used directly to providetherapeutic benefits in the treatment of cancers and other diseases. Asillustrated in the examples below, the compositions of the inventionhave been shown to induce degradation in Her2 kinase in SKBr3, MCF-7 andBT474 breast cancer cells. In addition, compositions of the inventionhave been shown to be effective at causing Rb-positive SKBr3 and MCF-7breast cancer cell lines to undergo G1 arrest, and to haveantiproliferative effects against these cell lines and BT474, MDA-MB468and prostate cancer cell lines TSUPr and LNCaP.

The compounds of the PU family were tested for binding to Hsp90,degradation of Her2 total protein and for their antiproliferativeeffect. Table 1 summarizes the influence of the 9-N chain on thisactivity. Compounds of the PU family (FIG. 6) not listed in the tablewere either inactive or insoluble.

Compounds of the PU family were modified by addition of a fluorine groupas substituent X3 (C-2 fluorination) using the reaction protocol of FIG.7B. The results are summarized in Table 2. As shown, introduction of afluorine substituent increased the activity of compositions with commonX1 groups, and enhanced the activity/solubility of compositions with X1substituents for which activity was not reported in Table 1.

Variations in activity were also observed when additional halogensubstituents were added to the phenyl ring using the reaction schemeshown in FIG. 8B. As shown in Table 3, introduction of a halide at one,but not both of the positions not occupied by methoxy groups in PU3 ledto an increase in activity relative to PU3.

Compounds were prepared with the chlorine substituent as in compoundPU3PhCl and the fluorine substituent as in PU24F or PU29F. As shown inTable 4, these compounds were the most active of those tested so far.

Another location of potential variation in the PU family of molecules isin the length and character of the bridge between the purine and thephenyl groups. FIG. 10 shows a synthetic scheme for achieving suchvariation. Compound 69 was tested and was found to have weak binding tohsp 90-beta, but no detectable binding to hsp 90-alpha. This is thereverse of the binding selectivity observed for Compound 18. Thisprovides for selectivity between these two proteins.

As an alternative to the use of the compositions of the inventionindividually, coupled-compositions in which the hsp-binding nucleus asshown in general structures in FIG. 5 are derivatized by coupling totargeting moiety selected to specifically bind to a protein, receptor ormarker found on a target population of cells or it may be dimerized.(See FIG. 11) The targeting moiety may be a hormone, hormone analog,protein receptor- or marker-specific antibody or any other ligand thatspecifically binds to a target of interest. Particular targetingmoieties bind to steroid receptors, including estrogen and androgen andprogesterone receptors, and transmembrane tyrosine kinases, src-relatedtyrosine kinases, raf kinases and PI-3 kinases. Specific tyrosinekinases include HER-2 receptors and other members of the epidermalgrowth factor (EGF) receptor family, and insulin and insulin-like growthfactor receptors. Examples of specific targeting moieties includeestrogen, estradiol, progestin, testoterone, tamoxifen and wortmannin.Targeting moieties may also be antibodies which bind specifically toreceptors, for example antibodies which bind to Her2 receptors asdisclosed in International Patent Publications Nos. WO96/32480,WO96/40789 and WO97/04801, which are incorporated herein by reference.

Because of their ability to bring about the degradation of proteinswhich are essential to cellular function, and hence to retard growthand/or promote cell death, the hsp-binding compounds of the invention,with or without a targeting moiety, can be used in the therapeutictreatment of a variety of disease conditions. A suitable therapeutic isone which degrades a kinase or protein that is found in enhanced amountsor is mutated in disease-associated cells, or on which the viability ofsuch cells depends. The general role of HSP90 proteins in maintainingmalignancy in most cancer cells points to the importance of this targetin the development of anticancer agents. Thus, the therapeutic smallmolecules of the invention provide a novel modality for the treatment ofall cancers that require or are facilitated by an HSP90 protein. Forexample, the compositions of the invention can be used in the treatmentof a variety of forms of cancer, particularly those that overexpressHer2 or mutated or wild type steroid receptors, or that lack functionalRB protein. Such cancers may include but are not limited to breastcancer and prostate cancer. In addition, the compositions of theinvention can be used in the treatment of other diseases by targetingproteins associated with pathogenesis for selective degradation.Examples of such targetable proteins include antigens associated withautoimmune diseases and pathogenic proteins associated with Alzheimer'sdisease.

The compositions of the invention are suitably utilized to degradespecific proteins which are associated with the disease state orcondition of concern. Because the compositions of the invention can beselected to degrade specific kinases or signaling proteins, a suitabletherapeutic is one which degrades a kinase or protein that is found inenhanced amounts in diseased cells. Thus, for example, the selection ofa binding moiety which degrades Her2 kinase, but not other HER kinasesor Raf kinase would be suitable for treatment of Her2-positive breastcancer. The examples below provide guidance on the selection of specificcompounds based on the specificity observed in vitro. In addition,screening techniques are described below to facilitate the evaluation ofstructures for binding affinity and differential degradation.

The compositions of the invention are administered to subjects,including human patients, in need of treatment, in an amount effectiveto bring about the desired therapeutic result. A suitable administrationroute is intravenous administration, which is now commonly employed inchemotherapy. In addition, because the compositions of the inventionsare small soluble molecules, they are suitable for oral administration.The ability to use an oral route of administration is particularlydesirable where it may be necessary to provide treatment of a frequent,for example a daily schedule. The amount of any given composition to beadministered, and the appropriate schedule for administration aredetermined using toxicity tests and clinical trials of standard design,and will represent the conclusion drawn from a risk benefit analysis.Such analyses are routinely performed by persons skilled in the art, anddo not involve undue experimentation.

As an alternative or adjunct to the design of molecules for use asbinding moieties in accordance with the invention, we have developed afast screening assay which can be used to test compounds for theirbinding affinity to the N-terminal pocket of HSP90, or to test proteinsfor the presence of an HSP90 type of pocket. As summarized in FIG. 12,the surface of a solid substrate (such as a the bottoms of 96 wellmicrotiter plates) are functionalized with N-oxysuccinimydylfunctionalities. These react in isopropanol with amino-functionalized GM(or other ansamycin antibiotic). The amount of GM bound to the wells canbe assessed by spectrophotometric absorbance measurements at 345 nm.When the bound GM is incubated with rabbit reticulocyte lysates, enoughhsp90 is captured to be detectable by colorimetric methods, althoughmore specific detection methods are preferred. For example, bound hsp90can be detected using a labeled anti-HRP antibody. To assay for HSP90binding efficacy, a test compound is added to the wells with the rabbitreticulocyte lysate (or other hsp90 source) and any differences in theamount of captured hsp are noted. If the test compound binds hsp90 withgreater affinity than GM, it will compete with the immobilized GM andresult in a reduction in the amount of hsp90 captured. By varying themember of the HSP90 family used in the assay, these same plates can beused to evaluate differences in specificity of test compounds. Theplates can also be used to screen protein/peptide libraries for proteinswhich possess an HSP90 type of binding pocket.

A test was also designed which allows identification of compounds whichinteract differentially with Hsp90-alpha and Hsp90-beta. In this test,geldanamycin (or other strong non-discriminating hsp90 binder such asother ansamycin antibiotics or radicicol) is modified as necessary andaffixed to a solid support, for example beads. An Hsp90 proteinpreparation containing both the alpha and the beta form is incubatedwith the support in the presence or absence of a compound to beevaluated. If the compound binds to the Hsp90 protein, it competitivelyinhibits the binding of the protein to the solid support. After washing,the material bound to the support is eluted and the eluate is separatedon an SDS/PAGE gel and visualized by immunoblotting withanti-Hsp90-alpha and anti-Hsp90-beta to determine the amount of materialbound. Alternatively, if quantitative amounts of the Hsp90 proteinpreparation or known ratios of alpha to beta forms of Hsp90 are used inthe first instance, the unbound material can be analyzed by a similartechnique.

Candidate compounds in accordance with the invention can also beevaluated for their ability to deplete or induce proteins which arefound in enhanced amounts in cancer cells or on which the cells dependfor viability (for example Her2, Her3, Raf-1, ER, Rb, cdk-4, Hsp90,Trap-1, and Grp94) in a panel of cancer cell lines using the cell-basedassay of Stockwell et al., Chem Biol. 6: 71-83 (1999). In this assay,cells are grown at the bottom of a well and fixed. The fixed cells areprobed for the presence of a specific primary antigen (the oncogenicprotein) using a specific primary antibody in solution. A secondaryantibody covalently linked to horseradish peroxidase is added, and theresulting complex is detected through the chemiluminescent reactioncaused by the addition of luminol, hydrogen peroxide and an enhancersuch as p-iodophenol. Differences in the detected luminescence levels inthe presence of a drug indicate activity of that drug with respect tothe degradation or induction of the targeted protein.

Drugs may also be assayed based upon observed very characteristicchanges in cell morphology. MCF-7 cells exposed to PU3 flatten, increasein size and have distinct cellular boundaries. The increase in size ismostly due to an abundance in cytoplasm. These morphological changes arecharacteristic of mature epithelial differentiation and reversal oftransformation.

As a third alternative, Immunofluorescence (IF) and Hematoxylin andEosin stain (H&E) may be used to assess drug effectiveness. Cells wereplated on 8 well chamber slides, Fisher Scientific) and seeded for 24hrs. Drugs or vehicle were added for 5 days after which, for IF theslides were washed twice with ice-cold PBS and fixed with methanol andacetone solution (1:1) for 15 sec. Fixed monolayers were washed withdistilled water and blocked with 5% BSA in PBS solution. After blocking,cells were incubated with the primary antibody (anti-MFMG, Chemicon,1:100 in 5% BSA in PBS) at 37° C. and washed 3 times with 1% BSA in PBS,followed by incubation with a rhodamine-labeled secondary antibody for 1hr at 37° C. Nuclei were stained with DAPI at 1 mg/ml. For H&E, thecells were fixed with paraformaldehyde (4%) for 10 min at RT and stainedaccording to standard H&E staining protocols. The induction of G1 arrestby a variety of manipulations is sufficient for induction of expressionof some milk proteins. However, only ansamycin, the HDAC inhibitor SAHAand the hsp90 binding molecules cause extensive morphological andbiological changes.

For use in the therapeutic method of the invention, the compositions ofthe invention are formulated in a pharmaceutically acceptable carrier.For injectable formulations, this may be a sterile solution (for examplesterile saline), or the compounds may be formulated in a lipidiccarrier. For oral formulations, the compositions of the invention may bepackaged in convenient dosage unit form, for example as tablets orcapsules with suitable excipients, or as a liquid formulation. In thelatter case, the liquid pharmaceutical will suitably include a flavorantto enhance palatability, and may also include colorants and otherconventional additives.

EXAMPLE 1

Compositions having general formula 1 were synthesized according to thescheme shown in FIG. 6A. To generate acid fluorides, to a solution ofthe phenyl acetic acid derivative (3 mmol) in 15 mL of CH₂Cl₂ (underinert atmosphere) were added 1.5 equivalents of cyanuric fluoride andpyridine (3 mmol). The mix was stirred for 1.5 h at room temperature,after which, 30 more mL of CH₂Cl₂ were added. The resulting solution waswashed once with 0.5 mL water, and the crude acid flouride materialresulted from the removal of the solvent was used in the next step.

The acid fluoride was used for the generation of PY-A, PY-B and PY-Cderivatives as follows: To 675 mg (2.8 mmol) of 4,5,6-triaminopyrimidinesulphate in 1.5 mL of DMF were added 1.5 mL (9 mmol) of DIEA, the acidfluoride (obtained as described above) dissolved in 5 mL CH₂Cl₂ and acatalytic amount of DMAP (0.28 mmol). The reaction mixture was stirredunder argon for 1 hr. The solid that formed was filtered off and thefiltrate was concentrate to dryness. To the residue material was added10 mL EtOAc and the precipitate formed was washed several times withEtOAc and CH₂Cl₂ to give a slightly yellow material in 60-80% yield.This material was used for the next step. If higher purity is desired,flash chromatography using EtOAc: MeOH=7:1 (1% TEA) can be performed.

From the PY compounds, PU-A, PU-B and PU-C derivatives were generated asfollows: To 500 mg (1.5 mmol) of 5-acylated-4,5,6-pyrimidine in 13 mLMeOH was added 13 mL solution of 25% NaOMe in MeOH and the resultingsolution was refluxed for 5 hrs. After cooling, 5 mL of water was addedand the solution was concentrated to 5 mL. The resulting aqueoussolution was extracted with 4×10 mL THF and 2×10 mL EtOAc. The combinedorganic layers were dried with MgSO₄, concentrated to dryness to give aslightly yellow solid in 80-90% yield. If higher purity is desired theresulting product can be added to a silica gel column and eluted withCH₂Cl₂:EtOAc:MeOH=4:4:1.

Substituents were added to the 9-N position using a Mitsunobu alkylationas follows: To 0.16 mmol of purine derivative in 5 mL toluene and 1 mLCH₂Cl₂ were added 2.2 equivalents PPh₃ and 1.3 equivalents of ROHfollowed by 5 equivalents of DEAD. The reaction was monitored by TLC andproceeded in 15 min to 1 hr. The mixture was applied to a ISCOCombiFlash system (silica gel column) and eluted with a gradient ofCH₂Cl₂/acetone to give 30-75% isolated product. This procedure wascarried out using the 40 unbranched and branched, linear and cyclic,saturated and unsaturated primary and secondary alcohols shown in FIG.6. In these syntheses, only two alcohols, neopentyl alcohol andcyclohexanol, were found to give no product in this reaction.

Four of the compounds (PU-A-4, PU-B-4, PU-C-4 and PU-C-15) were used ininitial tests. For convenience, these four structures are referred to asPU1, PU2, PU3 and PU4, respectively as shown in FIG. 13.

EXAMPLE 2

The human cancer cell lines MCF-7, SKBr3 and MDA-MB-468 were obtainedfrom the American Type Culture Collection (Manassas, Va.) and maintainedin 1:1 mixture of DME:F12 supplemented with 2 mM glutamine, 50 units/mLpenicillin, 50 units/mL streptomycin and 5% (for MCF-7 and MDA-MB-468)or 10% (for SKBr3) heat inactivated fetal bovine serum (GeminiBioproducts) and incubated at 37° C. in 5% CO₂.

For assays on protein levels, cells were grown to 60-70% confluenceexposed to drugs or DMSO vehicle for the indicated time periods. Lysateswere prepared using 50 mM Tris pH=7.4, 2% SDS and 10% glycerol lysisbuffer. Protein concentration was determined using the BCA kit (PierceChemical Co.), according to the manufacturers instructions. Clarifiedprotein lysates (20-50 mg) were electrophoretically resolved ondenaturing SDS-PAGE, transferred to nitrocellulose and probed with thefollowing primary antibodies: anti-Her2 (C-18), -Her3 (C-17), -Raf-1,-cyclin D1, -Rb (C-15) (Santa Cruz), anti-hsp90, -hsp70, -ER(Stressgen), anti-Trap-1 (MSK81), anti-b-actin, -tubulin (Sigma),anti-PI3K (p85) (Upstate Biotechnologies).

To determine antiproliferative indices, growth assays were performed byseeding 10,000 cells per well in 6-well dishes and incubating for 24 hrsbefore drug treatment. Drugs or vehicle were administered as outlinedfor each experiment, and cells were incubated for the time periodsdepicted and then counted by coulter counter.

Cell cycle distribution was assayed according to Nusse et al with aBecton Dickinson fluorescence activated cell sorter and analyzed by CellCycle Multi-cycle system (Phoenix Flow System, San Diego, Calif.).

Experiment 1

MCF-7 cells were treated with varying concentrations (0, 10, 50, 100 or250 μM of PU3 or the control purine Ad-But for 24 hours and then lysed.Levels of Hsp90, Trap1 and Hsp70 were evaluated by Western blotting. Theresults showed that PU3, like GM, increases the cellular levels of Hsp90and Hsp70. Treatment of cells with PU3 also induces a protein band thatmigrates more rapidly than Trap-1 and that is recognized by ananti-Trap-1 antibody. Although the identity of this protein band isunknown, its appearance seems to be a marker of cellular exposure toHsp90 inhibitors. Ad-But had no effect on the studied protein levels atidentical concentrations.

Experiment 2

Cell cultures of MCF-7, SKBr3 and MDA-MB-468 cells were treated with PU3at one of several concentrations or a DMSO control to test for theoccurrence of antiproliferative effects. FIGS. 14A-C show the results ofcell number as a function of time. As shown, PU3 clearly inhibits growthof these breast cancer cell lines.

Experiment 3

Cell cultures of SKBr3 cells were treated with PU1, PU2, PU3 or PU4 atconcentrations of 0, 10, 50, 100 and 250 μM. After 24 hours, the cellswere lysed and the amount of Her2 and Raf-1 in the cells was evaluated.Although the compounds are structurally very similar, they were found tohave different efficacy at promoting degradation of the two proteins.PU1 showed little degradation of either protein at any of theconcentrations tested, while PU2 degraded Her2 but not Raf-1 at 250 μM.PU3 degraded Her2 at concentrations over 100 μM but resulted in onlypartial loss of Raf-1 at 250 μM. PU4 degraded Her2 at concentrationsgreater than 50 μM and Raf-1 at a concentration of 250 μM.

Experiment 4

To test the time course of degradation, MCF-7 cells were treated with100 μM PU3. Aliquots of cells were recovered at 1.5, 3, 6, 12 and 24hours, and the amount of Her2, Raf-1, Hsp90 and Trap-1 was evaluated.The compound was shown to induce rapid degradation of Her2 (with morethan 50% being lost within 3 hours of adminstration) and significantsynthesis of Hsp90 and Hsp70 within the 24 hour period. The amount ofRaf-1 did not change significantly during this time.

Experiment 5

Cell cultures of MCF-7 cells were treated with PU1, PU2, PU3 or PU4 atconcentrations of 0, 10, 50, 100 and 250 μM. After 24 hours, the cellswere lysed and the amount of Her2 in the cells was evaluated. Althoughthe compounds are structurally very similar, they were found to havedifferent efficacy at promoting degradation of Her2. PU1 showed onlypartial degradation at concentrations in excess of 50 μM, while PU2degraded Her2 completely at 250 μM. PU3 substantially degraded Her2 atconcentrations over 10 μM. PU4 substantially degraded Her2 atconcentrations greater than 100 μM.

Experiment 6

Cell cultures of MCF-7 cells were treated with PU1, PU2, PU3 or PU4 atconcentrations of 0, 10, 50, 100 and 250 μM. After 24 hours, the cellswere lysed and the amount of estrogen receptor in the cells wasevaluated. Although the compounds are structurally very similar, theywere found to have different efficacy at promoting degradation ofestrogen receptors. PU1 showed essentially no degradation at theconcentrations tested, while PU2 degraded estrogen receptor completelyat 250 μM. PU3 partially degraded estrogen receptor at concentrationsover 50 μM. PU4 substantially degraded Her2 at concentrations greaterthan 100 μM.

Experiment 7

Cultures of SKBr3 and MCF-7 cells were treated with varyingconcentrations (0, 10, 50 100, or 250 μM) of PU3 for 24 hours. The cellswere lysed and levels of Her2, Raf1, Her3, estrogen receptor (ER), p85(PI3K), tubulin and actin were analyzed by western blotting. Reductionin the amount of those signaling proteins which depend on hsp90 fortheir function (Her2, Raf1, Her3 and ER) was observed. No effect wasobserved on PI3K, tubulin or actin, proteins which are involved in othersignaling pathways.

EXAMPLE 3

The experiments of Example 2 demonstrate the ability of syntheticcompounds PU1, PU2, PU3 and PU4 in accordance with the invention toprovide differential degradation of kinases HER3, estrogen receptor(ER), Her2, Raf, Rb and p85 (as reference protein which should not be,and is not degraded). Several important observations can be made fromthis data.

PU3 destabilizes the estrogen receptor complex and inducesdose-dependent degradation of the protein. PU3 also causes a rapiddecrease in Her2 levels and causes accumulation of a lower-molecularweight (170 kDa versus 180 kDa mature protein) HER-2 band, also seen forGM, that is thought to be incompletely glycosylated Her2 which issequestered in vivo in the endoplasmic reticulum. Levels of Raf and Her3are less sensitive to PU3, but are degraded at higher concentrations.PU2 also induces a lower molecular weight Rb band which is thought to behyperphosphorylated Rb.

In contrast, PU2 showed substantial degradation of Her2 withoutaccumulation of the lower molecular weight band and was less effectivefor degradation of Raf kinase. The lower molecular weight band is alsoabsent when PU1 is used as the binding moiety. PU2 is less effective atdegrading ER, while PU1 was had substantially no affect on ER. Thesedifferences provide evidence suggesting that PU3 binds to a differentmember of the HSP90 family from PU1 and PU2, thus accounting for thedifferent specificity. PU4 also shows greater ability to degrade Her2than ER or Raf.

EXAMPLE 4

Six compounds of the PU-C family (X4=H, X2=1, 2, 3 OMe, X3=H and withvariable X1 groups as indicated in FIG. 15) were prepared and tested fortheir effect on Her2, HER3 and TRAP-1 in MCF-7 cells. The cells weretreated for 24 hours with 10, 50, 100 and 250 μM of each compound. Thenature and size of the X1 group has a significant effect on the activityof the compound. Large, hydrophobic groups which will better fill theN-terminal pocket show the ability to degrade Her2, but not Her3. Inaddition, for these compounds there is a correlation between the abilityto degrade Her2 and induction of Trap-1 synthesis.

On the other hand, the compounds with big, rigid and somewhat polar X1groups interact less favorably, and do not significantly degrade Her2 atthe concentrations tested.

EXAMPLE 5

PU3 was linked to Biotin through the middle OH in accordance with thesynthetic scheme shown in FIGS. 16A and B. PU3-biotin was immobilized onSepharose-Streptavidin beads. These beads were used to show theinteraction of PU3 with the Trap-1 and Hsp90alpha

EXAMPLE 6

PU3 was tested for its safety and potency in MCF-7 xenografts. Thecompound was administered IP up to 500 mg/kg without showing significantsigns of toxicity. Mice with established tumors were treated with asingle dose of PU3 at doses of 50, 100 and 500 mg/kg i.p. Control micewere treated with DMSO alone. Mice were sacrificed 12 hourspost-treatment. For immunoblotting, tumor tissue was homogenized in 2%SDS lysis buffer (pH 7.4). In all cases a reduction in Her2 levels wasobserved in the immunoblotting results. No change was observed in levelsof PI3 kinase (p85).

EXAMPLE7

Several 9-Alkyl-8-benzyl-9H-purin-6-ylamines (4-46) were created by9-N-alkylation of PU-C (FIG. 6B). Additionally, the 2-amino group of 3(FIG. 7B) was converted to fluorine via a diazotization reaction innon-aqueous media using tert-butyl nitrite (TBN) in HF/pyridine. Theresulting purine was alkylated to give the2-fluoro-9-alkyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamines 47-59.The alkylation was performed using the Mitsunobu reaction, methodologythat can accommodate a large array of primary and secondary alcohols.The 2-fluoro derivatives were converted to the 2-alcoxy purines 60 and61 by refluxing in the corresponding alcohol and NaOMe. The majority ofmodifications at position 2 of the purine moiety commenced with (3).Iodine was introduced in that position using isoamyl nitrite indiiodomethane to give 62. The 2-iodo derivative was transformed to thecyano-derivative 63 with tri-n-butylcyanostannane andtetrakis(triphenylphosphine)palladium(0) in DMF and to thevinyl-derivative 64 with vinyltributyltin and (MeCN)₂PdCl₂.

The benzyl moiety was enriched in electron-donating groups in order toincrease its interaction with the pocket lysine. Chlorine and brominewere added via a radicalic reaction using t-butyl hydroperoxide and thecorresponding acid halide. In the case of chlorine only monosubstitutionwas observed (65), while bromine gave the monosubstituted (66) and asmall amount of di-bromosubstituted product (67). (FIG. 8B)

The nature and length of the bridge between the purine and the phenylring were additionally modified. As starting material we utilized the8-Br-adenine. This was reacted at high temperature with the aniline-,phenol- or benzyl-derivative to give the corresponding products 68-70.(FIG. 10)

The assembly of the fully substituted derivatives 71 and 72 commencedwith 3. Chlorine was added initially via the radicalic reactiondescribed above to give 73. Subsequently, the 2-amino group wastransformed to fluorine and the resulting purine (74) was alkylated viathe Mitsunobu reaction.

Specific reactions for various compounds are set forth below: 3 (DAAC):8-(3,4,5-Trimethoxy-benzyl)-9H-purine-2,6-diamine: To trimethoxyphenylacetic acid (1.0 g, 4.4 mmol) in DCM (20 mL) (under inert atmosphere)was added cyanuric fluoride (371 mL, 4.4 mmol) and pyridine (356 mL, 4.4mmol). The mixture was stirred for 1 h at room temperature, after which,an additional 30 mL of DCM was added. The resulting solution was washedonce with water (5 mL), and the acid fluoride resulted from the removalof the solvent was taken up in DMF (10 mL) and used in the next step.Separately, 2,4,5,6-tetraaminopyrimidine sulfate (0.9 g, 3.8 mmol) wasdissolved in water (40 mL) that contained NaOH (456 mg, 11.4 mmol). Theresulting solution was heated to 70° C. and the acid fluoride was addeddrop wise over a 20 minute period. The reaction mixture was stirred for1.5 h at 70° C. and then concentrated to dryness. To the crude materialwas added MeOH (16 mL) and a 25% solution of NaOMe in MeOH (16 mL) andthe resulting solution was heated at 90° C. for 18 h. Following cooling,the pH of the solution was adjusted to 7 by addition of concentratedHCl. The aqueous solution was removed and the crude taken up in DCM (100mL) and MeOH (50 mL). The undissolved solids were filtered off and theproduct (470 mg, 37%) was purified on a silica gel column withEtOAc:DCM:MeOH at 4:4:2.

FAC: 2-Fluoro-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine: To 3 (500mg, 1.5 mmol) was slowly added a 70% solution of HF in pyridine (2 mL),pyridine (8 mL), followed by t-butyl nitrite (200 mL, 2.0 mmol). Themixture was stirred for 2 h and then quenched overnight with 8 g CaCO₃in water (15 mL) and MeOH (10 mL). The solution was concentrated todryness and the resulting crude was taken up in MeOH (30 mL) and DCM (10mL). The insoluble solids were filtered off and the solvent was removedto give the crude product. This was purified on a silica gel columneluting with hexane:DCM:EtOAc:MeOH at 10:5:5:0.75 (240 mg, 50%).

The alkylation was performed via a Mitsunobu reaction as describedbefore (ref). Essentially, to FAC in toluene:DCM at 5:1 was added 1.3eq. of the corresponding alcohol, 2 eq. PPh₃, 3 eq. di-tert-butylazodicarboxylate and the resulting solution was stirred at roomtemperature for 10 min to 1 h (conversion was monitored by TLC) to give:

-   48 (PU3F):    2-Fluoro-9-butyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine: 60%    yield.-   52 (PU29F):    2-Fluoro-9-(2-isopropoxy-ethyl)-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine    86% yield.-   54 (PU47F):    2-Fluoro-9-pent-4-enyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    86% yield.-   59 (PU20F):    2-Fluoro-9-(tetrahydrofuran-2-ylmethyl)-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    53% yield.-   50 (PU44F):    2-Fluoro-9-(2-methoxy-ethyl)-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    28% yield.-   47 (PU43F):    2-Fluoro-9-propyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    66% yield.-   57 (PU24F):    2-Fluoro-9-pent-4-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    76% yield.-   53 (PU8F):    2-Fluoro-9-but-3-enyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    42% yield.-   55 (PU48F):    2-Fluoro-9-but-3-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    36% yield.-   56 (PU49F):    2-Fluoro-9-pent-3-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    25% yield.-   58 (PU16F):    2-Fluoro-9-cyclobutylmethyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    33% yield.-   51 (PU26F):    2-Fluoro-9-[(S)-2-methyl-butyl]-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    68% yield.-   49 (PU21F):    2-Fluoro-9-pentyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    65% yield.-   PU24DA:    9-Pent-4-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-purine-2,6-diamine: To    DAAC (200 mg, 0.61 mmol) of in toluene (20 mL) and DCM (4 mL) was    added PPh₃ (330 mg, 1.3 mmol), 4-pentyne-1-ol (75 mL, 0.8 mmol) and    di-t-butyl azodicarboxylate (600 mg, 2.5 mmol). The mixture was    stirred at room temperature for 2 h. The product was isolated by    column chromatography on a silica gel column eluting with    hexane:CHCl₃:EtOAc:EtOH at 10:8:4:4 (120 mg solid, 49%).-   62 (PU24I):    2-Iodo-9-pent-4-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    To PU24DA (50 mg, 0.13 mmol) was added CH₂I₂ (2.5 mL) and isoamyl    nitrite (100 mL, 0.78 mmol) and the resulting solution was heated at    80° C. for 1 h. After cooling, the solution was concentrated and    then added to a silica gel column. The product was isolated (40 mg,    61%) using eluent hexane:CHCl₃:EtOAc:EtOH at 10:4:4:0.75.-   63 (PU24CN):    2-Cyano-9-pent-4-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:    A solution of PU24I (20 mg, 0.04 mmol), Pd(PPh₃)₄(7 mg, 6.3×10⁻³    mmol) and tributyltin cyanide (14 mg, 0.043 mmol) in dry DMF (2.5    mL) was heated at 180° C. for 6 h. Following cooling and removal of    the solvent, the product (13 mg, 81%) was isolated by column    chromatography (hexane:CHCl₃:EtOAc:EtOH at 10:8:4:0.75).-   64 (PU24V):    9-Pent-4-ynyl-8-(3,4,5-trimethyl-benzyl)-2-vinyl-9H-purin-6-ylamine:    A solution of PU24I (20 mg, 0.04 mmol), (MeCN)₂PdCl₂ (2×10⁻³ mmol)    and tributylvinyltin (12.5 mL, 0.041 mmol) in dry DMF (3 mL) was    heated at 100° C. for 1 h. Following cooling and removal of the    solvent, the product (15 mg, 92%) was isolated by column    chromatography (hexane:CHCl₃:EtOAc:EtOH at 10:8:4:1).-   60 (PU3OMe):    2-Methoxy-9-butyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine: To    a solution of PU3F (20 mg, 0.051 mmol) in MeOH (1 mL) was added a    25% solution of NaOMe in MeOH (1.5 mL). The resulting mixture was    heated at 85° C. for 2 h. Subsequent to cooling, the mixture was    neutralized with 4 N HCl and then concentrated to dryness. The crude    was purified on a silica gel column with hexane:EtOAc:DCM:MeOH at    10:5:5:1 to give a solid (11 mg, 51%).-   61 (PU3OEt):    2-Ethoxy-9-butyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine: To    a solution of PU3F (20 mg, 0.051 mmol) in EtOH (2 mL) was added    NaOMe (15 mg, 0.28 mmol) and the mixture was refluxed for 2 h. After    cooling and neutralization with HCl, the solution was concentrated    to dryness. The product (12 mg, 56%) was purified as described    above.-   65 (PU3Cl):    9-butyl-8-(2-chloro-3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine: To    PU3 (37 mg, 0.1 mmol) in MeOH (3 mL) was added concentrated HCl (33    mL, 0.4 mmol). The solution was cooled to 0° C. and a 90% aqueous    solution of t-butyl hydroperoxide (44 mL, 0.4 mmol) was added. The    resulting solution was refluxed overnight. Following cooling and    removal of the solvent, the product (31 mg solid, 72%) was isolated    by column chromatography (elute with hexane:EtOAC:DCM:MeOH at    10:5:5:1.5).-   66 and 67 (PU3PhBr and PU3PhBr2): To PU3 (37 mg, 0.1 mmol) in MeOH    (3 mL) was added a 48% aqueous solution of HBr (45 mL, 0.4 mmol).    The mixture was cooled to 0° C., and t-butyl hydroperoxide (44 mL,    0.4 mmol) was added dropwise. The resulting solution was stirred for    30 minutes at 0° C. and then refluxed for an additional hour.    Following cooling and removal of the solvent, the mixture was    separated by column chromatography (elute with hexane:EtOAC:DCM:MeOH    at 10:5:5:1.5) to give predominantly monobrominated product (31 mg,    69%) and a trace of dibrominated product (2.3 mg, 4.4%). 66 (PU3Br):    9-butyl-8-(2-bromo-3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine; 67    (PU3Br2):    9-butyl-8-(2,6-dibromo-3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine.-   73 (DAACPhCl): To a slurry of DAAC (1 g, 3.0 mmol) in MeOH (50 mL)    was added concentrated HCl (1.5 mL, 18 mmol). The resulting solution    was cooled to 0° C. and a 70% aqueous solution of t-butyl    hydroperoxide (2.5 mL, 8 mmol) was slowly added. The mixture was    stirred for 30 min at 0° C. and then refluxed for 20 h. The solvent    was removed under high vacuum to give clean product (1.09 g, 98%).-   74 (FDAACPhCl): To a 20% solution of HF in pyridine (6.5 mL) was    added (under inert atmosphere) pyridine (13.5 ml), followed by    DAACPhCl (1 g, 2.7 mmol). The mixture was stirred for 5 min and    consequently, t-butyl nitrite (450 mL, 3.5 mmol) was slowly added.    Stirring continued for another 30 min. The reaction was quenched by    addition of CaCO₃ (16.5 g) in water (10 mL): MeOH (10 mL) and    stirring for 2 h. The solution was concentrated and the resulting    slurry was taken up in DCM (50 mL): MeOH (50 mL). The insoluble    solids were filtered off and washed with DCM:MeOH at 1:1 (2×25 mL).    Following solvent removal, the product was purified on a silica gel    column eluting with hexane:EtOAc:DCM:MeOH at 10:5:5:1 (370 mg solid,    37%).-   71 (PU24FCl):    2-Fluoro-9-pent-4-ynyl-8-(2-chloro-3,4,5-trimethoxy-benzyl)-9H-purin-6ylamine:    (308 mg, 76%).-   72 (PU29FCl):    2-Fluoro-9-(2-isopropoxy-ethyl)-8-(2-chloro-3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine:-   68 (PAM3):    9-Butyl-N*8*-(3,4,5-trimethoxy-phenyl)-9H-purine-6,8-diamine: A    mixture of BrAd3 (50 mg, 0.186 mmol) and trimethoxyaniline (120 mg,    0.65 mmol) was heated at 160° C. for 30 min. Following cooling, the    solid was taken up in DCM (20 mL) and MeOH (5 mL) and any insoluble    solids were filtered off. The product (57 mg solid, 83%) was    purified on a silica gel column eluting with DCM:EtOAc:MeOH at    7:4:1.-   70 (PU3OBn):    9-Butyl-8-(3,4,5-trimethoxy-benzyloxy)-9H-purin-6-ylamine: A    solution of benzyl alcohol (250 mL, 2.5 mmol) in 25% NaOMe in MeOH    (50 mL) was stirred for 5 min. Following the removal of methanol,    BrAd3 (26 mg, 0.096 mmol) and Cu powder (10 mg, 0.16 mmol) was    added, and the mixture was heated for 2 min at 180° C. The product    was purified on silica gel column with hexane:DCM:EtOAc:MeOH at    10:5:5:1 (5.5 mg, 14%).-   69 (PU3OPh):    9-Butyl-8-(3,4,5-trimethoxy-phenoxy)-9H-purin-6-ylamine. A mixture    of trimethoxyphenol (75 mg, 0.4 mmol), t-BuOK (34 mg, 0.3 mmol), Cu    powder (10 mg, 0.64 mmol) and BrAd3 (26 mg, 0.1 mmol) was heated for    1 h at 140° C. The product (13 mg solid, 35%) was isolated by column    chromatography (hexane:DCM:EtOAc:MeOH at 10:5:5:1).

EXAMPLE 8

The human cancer cell lines MCF-7, SKBr3 and MDA-MB-468 were obtainedfrom the American Type Culture Collection (Manassas, Va.) and maintainedin 1:1 mixture of DME:F12 supplemented with 2 mM glutamine, 50 units/mLpenicillin, 50 units/mL streptomycin and 5% (for MCF-7 and MDA-MB-468)or 10% (for SKBr3) heat inactivated fetal bovine serum (GeminiBioproducts) and incubated at 37° C. in 5% Co₂.

Protein Assays. Cells were grown to 60-70% confluence and exposed todrugs or DMSO vehicle for the indicated time periods. Lysates wereprepared using 50 mM Tris pH 7.4, 2% SDS and 10% glycerol lysis buffer.Protein concentration was determined using the BCA kit (Pierce ChemicalCo.), according to the manufacturers instructions. Clarified proteinlysates (20-50 mg) were electrophoretically resolved on denaturingSDS-PAGE, transferred to nitrocellulose and probed with the followingprimary antibodies: anti-Her2 (C-18), -Her3 (C-17), -Raf-1, -cyclin D1,-Rb (C-15) (Santa Cruz Biotechnology), anti-hsp90, -hsp70 (Stressgen)anti-Trap-1 (MSK81), anti-b-actin, -tubulin (Sigma), ER, anti-PI3K (p85)(Upstate Biotechnologies).Antiproliferative index. Growth assays were performed by seeding 10000cells MCF-7 and MDA-MB-468) and 20000 cells (SKBr3) per well in 6-welldishes and incubating for 24 hrs before drug treatment. Drugs or vehiclewere administered as outlined for each experiment, and cells wereincubated for the time periods depicted and then the number quantifiedby a coulter counter.Tissue Culture IC₅₀ Studies. Growth inhibition studies were performedusing the sulforhodamine B assay described before. Experiments wereperformed with BT-474, MDA-MB-468, MCF-7, and TSU-Pr1. Stock cultureswere grown in T-175's flask containing 30 mL of DME (HG, F-12,non-essential amino acids, and penicillin and streptomycin), withglutamine, and 10% FBS. TSU-Pr1 were grown in RPMI 1640 with glutamineand 10% FBS. Cells were dissociated with 0.05% trypsin and 0.02% EDTA inPBS without calcium and magnesium.Experimental cultures were plated in microtiter plates (Nunc) in 100 uLof growth medium at densities of 1000 cells per well, except for BT-474which were plated at densities of 3000 cells per well. One column ofwells was left without cells to serve as the blank control. Cells wereallowed to attach overnight (BT-474 were allowed to attach for 48hours). The following day, an additional 100 uL of growth medium wasadded to each well. Stock drug or DMSO was dissolved in growth medium attwice the desired initial concentration. Drug or DMSO was seriallydiluted at a 1:1 ratio in the microtiter plate and added to duplicatewells. After 72 hours of growth, the cell number in treated versuscontrol wells was estimated after treatment with 10% trichloroaceticacid and staining with 0.4% sulforhodamine B in 1% acetic acid. The IC₅₀is calculated as the drug concentration that inhibits cell growth by 50%compared with control growth.Her2 degradation. Total Protein Assays. Cells were grown to 60-70%confluence and exposed to drugs or DMSO vehicle for the indicated timeperiods. Lysates were prepared using 50 mM Tris pH 7.4, 2% SDS and 10%glycerol lysis buffer. Protein concentration was determined using theBCA kit (Pierce Chemical Co.), according to the manufacturersinstructions. Clarified protein lysates (20-50 μg) wereelectrophoretically resolved on denaturing SDS-PAGE, transferred tonitrocellulose and probed with the anti-Her2 primary antibody (C-18)(Santa Cruz Biotechnology).Binding studies. Solid phase competition assays. GM was immobilized onAffigel 10 resin (BioRad) as described². The GM-beads were washed withTEN buffer (50 mM Tris.HCl pH 7.4, 1 mM EDTA, 1% NP-40) containingprotease inhibitors and then blocked for 45 min at 4° C. with 0.5% BSAin TEN buffer. Hsp90 protein from Stressgen (SPP-770) was incubated withor without drugs for 17 min on ice. To each sample were added 20 μLGM-beads and the mixtures were rotated at 4° C. for 1 hr followed by 3washes with 500 μL ice cold TEN each. The GM-beads bound protein waseluted from the solid phase by heating in 65 μL 1×SDS. Samples wereportioned in a 20 μL aliquot for Hsp90 alpha analysis and a 40 μLaliquot for Hsp90 beta analysis, applied to a SDS/PAGE gel andvisualized by immunoblotting with Hsp90 alpha (Stressgen # SPA-840) andHsp90 beta (NeoMarkers#RB-118), respectively.

Compounds PU4-72 were tested for binding to Hsp90, degradation of Her2total protein and for their antiproliferative effect. The results aresummarized in Tables 1, 2, 3 and 4.

TABLE 1 Influence of the nature of the 9-N chain on activity EC₅₀ IC₅₀Hsp90 EC₅₀ IC₅₀ Her2/ IC₅₀ IC₅₀ α Hsp90 β MCF-7 MCF-7 BT-474 MDA-468PU13 4 70 75 PU22 5 80 118 PU43 6 6.6 10.8 47 50 54 PU3 7 15 13 50 55 61PU21 8 22.5 11.8 62 50 73 PU41 9 16.6 20.4 98 100 70 PU9 10 28 114 69 85PU14 11 62.3 52.1 160 >100 130 PU26 13 25.3 15.7 62 70 71 PU15 15 1216.2 75 70 70 PU30 16 111.3 47.8 111 120 PU16 17 17 32 46 60 68 PU4 18120 >100 70 50 PU23 22 13.3 9.5 47 50 55 PU7 23 17.6 75 64 70 80 PU8 244 10.6 41 30 73 PU11 25 51 66 PU24 26 2.6 1.5 24 20 41 PU25 36 82 PU4437 4.1 12.4 65 85 79 PU29 38 1.4 1.7 39 45 72 PU20 41 4.4 3.4 92 70 80*all other compounds from FIG. 6B were either inactive or insoluble

TABLE 2 The influence of C-2 fluorination on activity EC₅₀ EC₅₀ IC₅₀Hsp90 Hsp90 IC₅₀ Her2/ IC₅₀ IC₅₀ α β MCF-7 MCF-7 BT-474 MDA-468 PU43F 474.1 5.3 25 30 30 25 PU3F 48 6 3.5 24 25 29 30 PU21F 49 22 8.9 36 35 4319 PU44F 50 7.9 13.8 64 65 60 35 PU26F 51 22 15 44 40 45 28 PU29F 52 21.3 16 15 16 PU8F 53 5 9.8 33 30 35 25 PU47F 54 10.7 9.9 45 50 46 33PU48F 55 10 3.5 25 20 28 16 PU49F 56 15 10.4 37 45 33 45 PU24F 57 6.20.7 11 5 14 15 PU16F 58 30.7 9.2 41 40 31 21 PU20F 59 2.3 6.8 23 20 2521Addition of either CN, vinyl, iodine, methoxy, ethoxy, NH₂ at position 2of the purine moiety decreased or abolished activity.

TABLE 3 The influence of introduction of an electron-donor group on thephenyl moiety EC₅₀ EC₅₀ IC₅₀ IC₅₀ Hsp90 Hsp90 IC₅₀ Her2/ IC₅₀ MDA- α βMCF-7 MCF-7 BT-474 468 PU3 7 15 13 50 55 61 PU3PhCl 65 18.6 4.6 19 25 3036 PU3PhBr 66 35.3 11.7 25 50 60 INSOL PU3PhBr2 67 >100 >100 30 >100 21Assimilation of the best substituents resulted in the derivatives 71 and72 (Table 4).

TABLE 4 PU24FCl 71 0.55 0.45 2 2 4.5 3 PU29FCl 72 0.65 0.52 5.4 3 4.54.5

EXAMPLE 9

The ability of PU3 and PU24FCl to induce degradation of Akt protein. Asshown in FIGS. 17A and B, the 30-fold difference in activity which wasobserved for Her2 degradation was also reflected in degradation of Akt.An approximately 30-fold difference was also seen in the Hsp90 bindingaffinity.

EXAMPLE 10

Data was analyzed to see if there was a correlation betweenconcentrations of PU-series compounds effective for inducing Her2degradation and the concentrations required for growth inhibition in theMCF-7 cell line, and between the Hsp90 binding constant and theconcentrations required for growth inhibition. As shown in FIGS. 18A andB, the experimental results suggest a good correlation between growtharrest, Her2 total protein degradation and Hsp90 binding efficacy.

EXAMPLE 11

Cell culture. The human cancer cell lines MCF-7, SKBr3 and BT-474 wereobtained from the American Type Culture Collection (Manassas, Va.) andmaintained in 1:1 mixture of DME:F12 supplemented with 2 mM glutamine,50 units/mL penicillin, 50 units/mL streptomycin and 5% (for MCF-7) or10% (for SKBr3 and BT-474) heat inactivated fetal bovine serum (GeminiBioproducts) and incubated at 37° C. in 5% CO₂.Her2 degradation. Total Protein Assays. Cells were grown to 60-70%confluence and exposed to drugs or DMSO vehicle for the indicated timeperiods. Lysates were prepared using 50 mM Tris pH 7.4, 2% SDS and 10%glycerol lysis buffer. Protein concentration was determined using theBCA kit (Pierce Chemical Co.), according to the manufacturersinstructions. Clarified protein lysates (20-50 μg) wereelectrophoretically resolved on denaturing SDS-PAGE, transferred tonitrocellulose and probed with the anti-Her2 primary antibody (C-18)(Santa Cruz Biotechnology). As shown in FIG. 19, PU24FCl inducesefficient degradation of the oncogenic protein Her2 at lowconcentrations (IC50 of 1.7, 2, 4.5 uM for SKBr3, MCF-7 and BT-474respectively).

1. A compound of the formula:

wherein Y is CH, O, N or O—CH, X₁ is the substituent formed by removingthe OH from an alcohol selected from the group consisting of

X₂ is from one to five non-hydrogen groups independently selected fromthe group consisting of halogen and methoxy, X₃ is halogen, X₄ is absentwhen Y is O, or X₄ is hydrogen, halogen, alkyl, alkoxy, —SCH₃ or—SCH₂CH₃, and X₆ is —NH₂, —OH, —O-Alkyl, or —CONH₂.
 2. The compoundaccording to claim 1, wherein X₂ is 1, 2, 3 trimethoxy.
 3. The compoundof claim 1, wherein X₆ is —NH₂.
 4. The compound according to claim 3,wherein X₂ is 1, 2, 3 trimethoxy.
 5. The compound of claim 1, having thestructure

wherein Me is methyl, and X₇ is hydrogen or halogen.
 6. The compound ofclaim 1, having the structure

wherein Me is methyl.
 7. The compound of claim 1 having the structure

wherein Me is methyl.